Elucidation of the mechanisms of hormone action in the mammary gland of the mouse constitutes the objective of this proposal. Prolactin and a corticosteroid (glucocorticoid) are required for production of casein, and regulation of this major milk-protein is often used as a measure of hormone-inducible, gene mediated differentiation of the breast cells. The discrete role(s) of prolactin and the glucocorticoid regulating milk-protein, as yet, has remained obsure, principally because of the inadequacies of the in vivo or the tissue fragment culture models used in most previous studies. We have developed a two step organ culture model in which the whole mammary gland sequentially mimics mammo genesis and lactogenesis in a chemically defined medium containing appropriate hormones. Abundant casein and its mRNA are produced in the gland, exclusively at 2nd step of cultivation, in medium containing prolactin and glucocorticoid. This two step organ culture model will be used to assess the precise mode of actions of prolactin and the glucocorticoid in regulation of the milk-protein and mammary cell differentiation. The mode of action of the two lactogenic hormones in the potentially tumorigenic, hyperplastic alveolar nodules (HAN) of the mammary gland will be assessed during cultivation of the HAN in a similar culture model. The relationship between hormone-receptor interactions and functional responses of the mammary cells also will be measured. The procedures used will include, monitoring of: (a) specific casein mRNA transcription in isolated nuclei, (b) cell-free translational activity of casein mRNA, and (c) RNA-cDNA (to casein mRNA) hybridization. Radioactively labeled hormones in ligand-receptor assays will be used for measuring prolactin and the glucocorticoid receptors. The results should substantially advance our understanding of the mode of hormone action during normal and neoplastic development of the breast cells.